The invention relates to a method of using DNA sequences for assessing seed purity. More specifically, the invention relates to a DNA sequences having homology to rice mitochondrial DNA and being unique to Wild Abortive (WA) cytoplasm containing cytoplasmic male sterile lines of rice, and the use of these sequences in a Polymerase Chain Reaction (PCR) assay to distinguish Male Sterile (CMS) lines of rice from their cognate Male Fertile Maintainer Lines. This invention relates to a method for ensuring the purity of cytoplasmic male sterile lines of rice using DNA based markers
Hybrid vigor is the phenomenon by which the progeny of a cross between two inbred lines has higher yield potential than either one of the parents. Hybrids can yield upto 10-30% more than the best variety and are a favored option for increasing yield.
Rice is a major cereal crop all over the world; and in many parts of Asia it is the staple part of the diet. It has been estimated that in a number of Asian countries like India rice yields must double by the year 2025 to meet the demands of the increasing population (Hossain, 1996. In Khush (ed) Rice Genetics III, Proc. Third Intl. Rice Genet. Symp., Los Banos Manila, the Philippines. Oct. 16-20, 1995. International Rice Rearch Institute, Manila, the Philippines). As has been demonstrated in the People""s Republic of China, where almost fifty percent of the area under rice cultivation is covered by hybrids, the widespread cultivation of hybrid rice is a readily available option for increasing yield. By comparison, in countries like India the area under hybrid rice is less than 1% of the total area under rice cultivation. This illustrates the tremendous potential for increasing the area under hybrid rice cultivation and it is expected that the market for hybrid rice seeds will increase in a number of rice growing countries, including in India.
The most widely used system for hybrid rice production is the three line system (FIG. 1). The three lines include: 1. a male sterile, female fertile line called the Cytoplasmic Male Sterile (CMS) line because it carries a male sterility conferring mutation in the cytoplasmic component of the genome, 2. a maintainer line and 3. a restorer line; the maintainer and restorer lines are male fertile as well as female fertile. The CMS and Maintainer lines are practically identical with respect to the nuclear component of the genome (and are often referred to as iso-nuclear lines) but differ from each other with respect to the cytoplasmic component of the genome. The male sterility of the CMS line is maternally inherited and is most likely due to a mutation in the mitochondrial DNA. The CMS line, being female fertile, can be propagated by fertilization with pollen emanating from the Maintainer line. Since the cytoplasmic component of the genome is not transferred through pollen, the progeny of such a cross would inherit the cytoplasm only from the CMS line and would therefore be male sterile. The nuclear component of the genome of the progeny would also be identical to that of the CMS line, even though half of it is inherited from the Maintainer line, as there is no difference between these two lines with respect to this component of the genome.
The hybrid seeds are produced in a cross of the CMS line with another inbred parental line, called the Restorer line, which as indicated above is Male fertile and Female fertile. In this cross the CMS line serves as the female parent while the Restorer line is the male parent. The Restorer line also carries a Rf (restorer of fertility) gene/s in it""s nuclear genome which will restore male fertility to a plant whose cytoplasm has been inherited from the CMS line. The hybrid seeds produced in the cross depicted in FIG. 1 would therefore be fertile. The CMS and Restorer lines are appropriately chosen such that the hybrids exhibit sufficient hybrid vigor (or heterosis) to produce substantially higher yields than inbred varieties.
The vast majority (90% or more) of the rice hybrids that are currently under commercial cultivation in the world derive their cytoplasm from a single source (Yuan, 1995. Hybrid rice seed production technology, Directorate of Rice Research, Hyderabad, India). This cytoplasm, called the WA (wild abortive) cytoplasm, was discovered in a wild rice plant in China. Subsequently, this cytoplasm has been crossed into several different nuclear genetic backgrounds by repeated back-crossing using the recurrent parent as the male donor. In this manner several different CMS lines have been developed, each of which has in turn been crossed with different Restorer lines to develop a number of hybrids, all of which share the same WA cytoplasm.
It is important to maintain the purity of hybrids as any impurities therein would reduce the expected yield. It has been estimated that for every one-percent impurity in the hybrid seed the yield reduction is to the tune of 100 Kg per hectare (Mao et al, 1996 In Virmani, S. S., E. A. Siddiq, and K. Muralidharan (eds) Advances in Hybrid Rice Technology. Proc. Third Intl. Symp. on Hybrid Rice, Directorate of Rice Research, Hyderabad, India). The Indian seed act prescribes that, for hybrid rice, the expected purity should be 98% (Verma, 1996. Seed Tech. News 24:1-4); in the People""s Republic of China it is mandated that the purity of hybrid rice should be at least 96% (Wengui Yan, 2000. U.S. Pat. No. 6,066,779). In order to ensure the required levels of hybrid seed purity, the parental lines that are employed in hybrid seed production should have a very high (almost 99%) level of purity.
One of the common admixtures that occur during hybrid seed production is that of Maintainer lines with those of the CMS lines. As these are iso-nuclear lines, it is very difficult to distinguish between these lines based on morphological criteria i.e. other than male sterility which can only be judged at the time of flowering. DNA markers that distinguish the CMS and maintainer lines can be developed and applied at the seedling level for the practical detection of seeds of Maintainer lines that occur as contaminants within stocks of the CMS lines. DNA Markers based on the use of the Polymerase Chain Reaction would be ideally suited for this purpose as they are much more efficient for handling large numbers of samples than hybridization based methods like Restriction Fragment Length Polymorphisms.
The Polymerase Chain Reaction is based on the use of short oligonucleotide sequences as primers for the enzymatic amplification of DNA sequences that occur between two appropriately spaced primer binding sites on the target DNA. The PCR works most reproducibly when the oligonucleotide primers are designed on the basis of a knowledge of the target DNA sequence. Protocols have been developed for PCR which are based on the use of short (8-10 bases long), randomly designed oligonucleotide primers (Williams et al, 1990; Nucleic acids research 18: 6531-6535). These primers are not based on a knowledge of the target DNA sequence and kits containing large numbers of these randomly generated primers are now commercially available. The DNA markers that are developed by this method are known as Randomly Amplified Polymorphic DNA (RAPD) Markers. Because a large number of primers are available, genetic polymorphisms can be detected by this method, even within closely related lines. However, the reproducibility of RAPD markers is poor due to the short length of the oligonucleotide (RAPD) primers and possibly also due to the lack of the required degree of specificity for the target. This severely limits the practical application of RAPD markers, as diagnostic markers for distinguishing different genotypes.
RAPD markers that distinguish CMS (WA cytoplasm) and Maintainer lines of rice have been described (Jena and Pandey 1999; Hybrid Rice Newsletter. 2: 13-14). However, the low reproducibility of these markers has made it practically impossible to apply them in a routine manner for distinguishing CMS and Maintainer lines. There is thus a need for the development of reproducible PCR based methods that can be applied for distinguishing the CMS and Maintainer lines. The desired level of reproducibility can be obtained if the oligonucleotide primers are based on a knowledge of the sequence of a region of rice DNA that is polymorphic between the CMS and Maintainer lines. PCR assays based on such primers would be highly reproducible because these primers would be longer in length than the primers used in RAPD analysis and would be specific for the target DNA sequence.
In this application, is described the identification and sequence determination of a region of rice mitochondrial DNA that is specific to CMS lines of rice containing the wild abortive (WA) type of cytoplasm. Based on this sequence, specific oligonucleotide primers have been developed that can be used in a PCR assay to distinguish CMS (WA) lines from their iso-nuclear Maintainer lines. These primers have been used to distinguish several different CMS lines (all containing the WA cytoplasm) of rice from their cognate Maintainer lines. In a coded test, this assay was used to predict with 100% accuracy the genotypes of a mixture of CMS (WA) and Maintainer lines of rice. The assay can therefore be used by rice breeders to successfully detect admixtures of Maintainer lines in seed stocks of the CMS line, thereby ensuring the purity of this parental line and the hybrid derived from it.
Another source of impurity within the seed stocks of the CMS line is caused by cross pollination with pollen emanating from rice plants that are not the designated Maintainer line. A minimum isolation distance of 300 meters is prescribed for multiplication of rice CMS lines (Virmani, 1993. Advances in Agronomy 57:377-462) i.e. within this distance no rice lines other than the Maintainer line (the preferred pollen donor or male parent) should be cultivated. This is based on the observation that pollen originating from rice plants growing beyond this distance will not pollinate the female parent. Occasionally, this minimum isolation distance is either not strictly followed or local conditions (for e.g. wind flow and weather) might permit pollen to be transferred from plants that are growing beyond the 300 meters distance. Therefore, it is. important to monitor the extent of outcrossing with rogue pollen donors that has occurred during the multiplication of the CMS lines. In this patent, are described methods for the application of sequence specific PCR markers like microsatellites and Sequence Tagged Sites (STSs) towards estimating the extent of outcrossing that has occurred during the multiplication of seed stocks of the CMS line.
Although the application is for estimating the extent of outcrossing that has occurred for CMS lines of rice, a similar approach can be used for estimating the extent of outcrossing that has occurred within the CMS lines of other crops including but not limited to maize, pearl millet, sorghum, wheat, sunflower, mustard, cabbage, caulifower, tomato, pepper, okra, etc wherein the CMS lines are used for the production of hybrids and appropriate microsatellite or STS markers are available.
The estimation of hybrid seed purity is conventionally done by the grow out test (GOT), which is based on the assessment of morphological and floral characteristics (that distinguish the hybrid) in a representative sample of plants that are grown to maturity. Rice plants take several months to reach maturity and the seeds have to be stored under appropriate conditions as they cannot be marketed until these results become available. In addition substantial delays can result, as occurs in India, if the first growing season after hybrid seed production which is taken up by the GOT is also the preferred season for hybrid cultivation. In such cases, the seeds have to be stored for upto a year i.e. until the subsequent growing season before they can be marketed. For seed companies, large amounts of capital are therefore locked up in the form of hybrid seed stock for prolonged periods while awaiting the results of the GOT. Another disadvantage of the GOT is that it can be subjective due to environmental influences on the expression of morphological characteristics. Further, there is also the possibility that adverse climatic conditions (like heavy wind or rain) can damage or destroy the crop and make it difficult to collect the data.
With the objective of replacing the GOT with a test that is superior in terms of speed and accuracy, a PCR based assay is described for assessing hybrid seed purity. This test involves the use of either microsatellite or STS (Sequence Tagged Site) polymorphisms that distinguish the parental lines of rice hybrids. These polymorphisms are co-dominant and the alleles are detected as DNA fragments of different sizes following PCR and agarose gel electrophoresis. The hybrid can be identified because it will have alleles contributed by both parents i.e. PCR amplified fragments of two different sizes will be obtained after use of the DNA isolated from the hybrid plant as a template in PCR. One of these alleles will be contributed by the male sterile, female fertile (CMS) parent while the other will be contributed by the male fertile, female fertile (Restorer) parent. This test can be conducted on DNA isolated from six day old rice seedlings and the assay can be completed within forty eight hours. The implementation of this PCR based test for seed purity will result in considerable savings for the seed industry. Additional modifications of this assay are described wherein the test need not be conducted on individual seedlings but can be conducted on populations of seedlings obtained from the hybrid seed stock.
This invention relates to novel DNA markers for assessing seed purity and a method for ensuring the purity of cytoplasmic male sterile lines of rice using DNA based markers. This method is based on the identification of a DNA Sequence that is specific to WA cytoplasmic male sterile lines of rice and the development of specific DNA markers derived from the same. These DNA markers can be used to detect admixtures of male fertile Maintainer lines with CMS lines. This application is likely to be very beneficial to the hybrid rice industry as admixtures of the type described above often lead to reduced purity of the hybrid seeds and poor performance of the product in the marketplace. Methodology for the application of co-dominant sequence specific PCR markers like microsatellites and STSs for detecting impurities in parental lines and hybrids of rice and other crops is also provided.
Rice is a major cereal crop in many parts of the world. Yield increases of 10-30% are reported following cultivation of hybrid rice in the People""s Republic of China where it is being practised on a large scale. It is expected that, in the near future, hybrid rice technology will also be practised on a large scale in a number of other rice growing countries. Currently, most rice hybrids are produced through a three line system comprising of: 1. a Cytoplasmic Male Sterile (CMS) line that is female fertile but is male sterile due to a mutation in the cytoplasmic (most probably the mitochondrial) component of the rice genome; 2. a male fertile, female fertile Maintainer line that is identical to the CMS line with respect to the nuclear component of the genome but has a different cytoplasmic genotype that does not induce male sterility; 3. a male fertile, female fertile Restorer line. The CMS line serves as the female parent for the hybrid while the Restorer line is the male parent. During hybrid seed production the CMS and Restorer lines are cultivated in close proximity to each other such that pollen emanating from the Restorer line will pollinate the flowers of the CMS line. As the CMS line is male sterile it will not set seed by self pollination and any seeds that are formed on the CMS line are deemed to have arisen as a consequence of fertilisation with pollen emanating from the Restorer line. The restorer line carries one or more nuclear encoded genes that will restore male fertility to the hybrid even though it carries the CMS cytoplasm. Thus the hybrid is self fertile.
The CMS line cannot be propagated by selfing as it is male sterile. Instead, the propagation of the CMS lines is accomplished by using the CMS line as a female parent and the Maintainer line as the male parent. The genotype of the progeny that arise from this cross will be identical to the genotype of the CMS line as the Maintainer lines are practically identical to the CMS line with respect to the nuclear component of the genome. The progeny will have the male sterile characteristic of the CMS line because the cytoplasmic component of the genotype is contributed by the female parent; which in this case is the CMS line. It is also pertinent to note that the Maintainer line does not carry any Restorers of the Cytoplasmic Male Sterile phenotype of the CMS line.
The identity of the nuclear genotype of the CMS and Maintainer lines means that the two lines are almost indistinguishable by morphological criteria. This creates a practical problem because it is very difficult to detect admixtures of the Maintainer line within seed stocks of the CMS line. Since the Maintainer lines are self fertile, these impurities can produce seeds in a hybrid rice production field without the necessity for fertilization with the Restorer line. This leads to a contamination of the seeds of the Maintainer line with those of the hybrid. This type of a contamination is one of the most frequently observed during hybrid rice seed production and leads to a reduction in the expected yield and poor performance of the hybrid in the field. If the purity of the hybrid is less than the mandatory limit fixed by Seed Certification agencies, this is 98% in India and 96% in China, the entire seed lot is rejected leading to considerable loss for the seed producers (companies).
The vast majority of the CMS lines that are employed in commercial production of hybrid rice are based on the use of the WA cytoplasm. In this patent we describe the identification of a DNA sequence that is unique to rice lines containing the WA cytoplasm and is highly homologous to rice mitochondrial DNA. Sequence Specific oligonucleotide primers have been developed based on this DNA sequence that can be used in a PCR assay to reliably distinguish rice cytoplasmic male sterile lines containing the WA cytoplasm from their cognate Maintainer lines. These primers can therefore be used by hybrid rice breeders/seed companies to detect impurities of the Maintainer line within the CMS line. By ensuring purity of the CMS lines, a major source of contamination of the hybrid seeds is removed leading to obvious benefits for the seed industry and farmers.